Fig. 4. Krm overexpression analysis. (A-G) Axis duplication assay performed by injection of indicated mRNAs into two opposite blastomeres at the four-cell stage. Amounts injected were 10 (Xwnt8), 200 (LRP6), 5 (Xdkk1) and 100 (mkrm2) pg per blastomere. (H-J) Both krm2 (I) and dkk1 (J) anteriorise Xenopus embryos, whereas preprolactin (ppl) control has no effect (H). mRNA (375 pg Xkrm2 or 50 pg Xdkk1 per blastomere) was injected into all blastomeres of four-cell stage embryos. (K) krm2 and dkk1 upregulate the anterior neural marker genes otx2 and XAG1 and the pan neural marker NCAM in animal cap RT-PCR assays. mRNA (500 pg of Xkrm2 and 200 pg Xdkk1) was injected in each blastomere of four-cell stage embryos. Actin was used to confirm absence of mesoderm in animal cap explants. -RT, minus reverse transcription control; H4, histone H4 used for RT-PCR sample normalisation. (L-N) krm2 blocks posteriorising Wnt activity. (M) 50 pg of pCSKA-Xwnt8 DNA injected into each animal blastomere of eight-cell stage embryos results in loss of head structures (70% headless, n=26). (N) Co-injecting 250 pg Xkrm2 mRNA with XWnt8 DNA completely rescues this phenotype (0% headless, n=46). (O-Q) krm2 rescues cyclopia induced by inhibitory anti-Dkk1 antibodies. mRNA [250pg of ppl (O,P) or krm2 (Q)] was injected into all blastomeres of four-cell stage embryos and the same embryos were then injected with either PBS (O) or 250 ng of anti-Dkk1 antibody at stage 9 (P,Q). Cyclopia as in P (65%, n=34) was completely rescued by krm2 injection (0%, n=40). Frontal views of embryos in O-Q are shown on the right.

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